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SIRT6 abrogation promotes adrenocortical carcinoma through activation of NF-KB signaling
Xueyi Wu1 . Haoming Tian11D . Long Xue2 . Lizhi Wang3
Received: 13 November 2018 / Accepted: 15 March 2019 @ Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
As an uncommon malignancy in the adrenal gland, adrenocortical carcinoma (ACC) is characterized by thorny diagnosis and poor clinical outcome, necessitating innovative treatment strategies. Sirtuin 6 (SIRT6), a tumor suppressor, modulates aerobic glycolysis of malignant cells and has an impact on tumorigenesis. This study focused on investigating SIRT6 expres- sion in ACC and how it generates cancer phenotypes. SIRT6 expression was inhibited in ACC tissues according to western blotting, real-time polymerase chain reaction, and immunohistochemistry. MTT assay, TUNEL assay, and flow cytometry were performed to evaluate the contribution of SIRT6 to cell invasion, proliferation, death, and migration. It was shown that SIRT6 knockdown promoted cell invasion, proliferation, and migration, and inhibited cell death. Moreover, it was found that SIRT6 knockdown upregulated TLR4 and reinforced phosphorylation of the nuclear transcription factor-kappa B (NF-KB) subunit p65 as well as inhibitor of nuclear factor kappa-B kinase. Additionally, SIRT6 knockdown significantly enhanced expression of calcitonin gene-related peptide as well as transient receptor potential vanilloid subtype 1. It also reinforced reactive oxygen species generation. Overall, our research findings demonstrate that SIRT6 serves as a tumor suppressor via regulation of the NF-KB pathway, which could offer an innovative strategy to treat ACC.
Keywords Sirtuin 6 · Adrenocortical carcinoma · NF-KB · Transient receptor potential vanilloid · Calcitonin gene-related peptide · Reactive oxygen species
Introduction
Adrenocortical carcinoma (ACC) is an uncommon but lethal malignancy with an incidence of 0.7-2.0 cases per million people per year, with a 5-year overall survival rate
☒ Haoming Tian hmtian9999@126.com
Xueyi Wu xueyiwu18@163.com Long Xue longhuaxiyiyuan@126.com Lizhi Wang lizhiwanghx88@126.com
1 Department of Endocrinology and Metabolism, West China Hospital, Sichuan University, No. 37, Guoxue Lane, Chengdu 610041, Sichuan, China
2 Department of Intensive Medicine, Women and Children’s Hospital of Sichuan Province, Chengdu 610043, China
3 Department of Eugenics, Women and Children’s Hospital of Sichuan Province, Chengdu 610043, China
of 37-47% [1]. Despite a few patients noticing symptoms due to hormonal hypersecretion and mass effects, ACC is incidentally and terminally discovered [2]. Surgery serves as the only possible curative option for patients suffering from ACC [3]. For ACC patients who cannot receive operations, adrenolytic as well as conventional chemotherapy exert mod- erate effects which are limited by their toxicity [4]. Although researchers have gone to great lengths to reveal ACC etiol- ogy, novel drugs have been unable to improve prognosis [5]. Treatment which targets vascular endothelial growth factor (VEGF) or insulin growth factor (IGF)-1 receptors has been unable to demonstrate improved survival [6, 7]. As a result, identification of innovative and effective targets for ACC treatment is urgently needed.
Transient receptor potential vanilloid subtype 1 (TRPV1), a ligand-gated ion channel, is expressed in nociceptor nerve terminals and plays a major role in detecting noxious stimuli. TRPV1 channel is the key molecular transducer of elevated temperature (>42 C) and associated with mechanical and thermal hyperalgesia after burn injury. Studies showed that TRPV1 induces the release of neuropeptides, substance P,
and calcitonin gene-related peptide (CGRP) and then pro- motes neurogenic inflammation in vivo. However, the role of TRPV1/CGRP signaling in ACC remains unknown.
As a part of sirtuin family of enzymes relying on NAD+, sirtuin 6 (SIRT6) serves as a deacylase relying on NAD+ as well as mono-ADP-ribosyltransferase in acetyl and long- chain fatty acyl groups [8]. SIRT6 participates in various biological functions, such as metabolism, transcription, DNA repair, inflammation, genome instability, aging, and telomere regulation as well as generation of malignancy [9-13]. It has been recently shown that SIRT6 serves as an oncogene or tumor suppressor in different human malignan- cies [14-16]. For instance, Fukuda et al. [17] demonstrated that SIRT6 hindered proliferation through stimulation of cell death in endometrial malignancies. Bai et al. [15] discovered that SIRT6 was an oncogene in NSCLC, which could pre- dict worse clinical outcome and severe metastasis through the ERK1/MMP9 pathway. Lin et al. [18] demonstrated that SIRT6 was downregulated in colon carcinoma, indicating that SIRT6 could be a tumor suppressor. Nevertheless, cur- rent knowledge of the contribution of SIRT6 to ACC as well as its mechanism is insufficient.
Consequently, our research aimed to investigate the expression as well as the impact of SIRT6 on ACC. We investigated impact of SIRT6 on ACC malignant cell migra- tion as well as viability. The findings of our research indicate that SIRT6 has potential for ACC treatment.
Materials and methods
Tissue samples
Eight tumor specimens were acquired from Tong Ji Hospital from June 2010 to September 2015. Normal samples were obtained via an experienced attending endocrine pathologist (TS) from tissue surrounding the malignant tissue or from adenoma or even normal adrenal glands as part of radical nephrectomies. This study has been approved by the Ethics Committee of the West China Hospital, Sichuan University. All study participants had given their written informed con- sent before participating in the study.
Immunohistochemistry (IHC)
Paraffin slices of colon samples were cut into sections with 4 um thickness. A 3-step IHC staining was carried out as mentioned before [24]: tissues were dewaxed and rehy- drated before a 10-min incubation with 3% H2O2 at 20 ℃. Tris-EDTA was used for antigen retrieval. Five percent goat serum in PBS was used for blocking. Samples were incu- bated overnight at 4 ℃ with anti-SIRT6 primary antibodies diluted to 1:100 (provided by Cell Signaling Technology,
America; No. 2590). Incubation with secondary antibod- ies was carried out before washing with PBS. Chromogen 3’-diaminobenzidine tetrachloride (provided by Serva, Ger- many) served as a substrate.
Cell culture
H295R (adrenocortical carcinoma cells) acquired from ATCC were preserved in a 1:1 mixture of Dulbecco’s modi- fied Eagle’s medium (DMEM) and Ham’s F12 medium. SW-13 cells were preserved with Leibovitz’s L-15 medium with 10% fetal bovine serum and 2 mM L-glutamine. Cul- tivation was carried out using humidified incubator at 5% CO2 and 37 °C.
RNA isolation and quantitative RT-PCR (qRT-PCR)
Trizol™M reagent (provided by Invitrogen) was utilized in order to isolate total RNA as per the manufacturer’s instruc- tions, which then underwent reverse transcription (RT) to generate cDNAs using a TIANscript RT Kit (purchased from TIANGEN, China). Expression was measured via qRT-PCR using SYBR master mix (supplied by Takara, China). GAPDH was the internal control. Expression was determined according to the 2-AACt method. Primers for PCR were as follows: SIRT6 F: GCA GTC TTC CAG TGT GGT GT; SIRT6 R: GAT CTG CAG CGA TGT ACC CA; GAPDH F: 5’-TGA CTT CAA CAG CGA CAC CCA-3’; and GAPDH R: 5’-CAC CCT GTT GCT GTA GCC AAA-3’.
Cell transfection
H295R cell line was transfected with SIRT6-specific siRNA or SIRT6 plasmids from Genomics Co (Beijing, China). The siRNA was added to 5 ul of water without RNase to a con- centration of 100 pmol/ul, which was subsequently mixed with 10 ul of transfection reagent and incubated for 15 min at 25 ℃. Forty-eight-hour transfection was carried out with siRNA.
ROS examination
ROS production in H295R cells was examined using a fluo- rescence probe, DCFH-DA (provided by Sigma, MO). Cells received a 30-min incubation with 10 umol of DCFH-DA at 37 ℃ under dark conditions. Fluorescence intensity was measured with an Olympus BX51 microscope.
MTT evaluation of cell proliferation
Cells (2×103 cells/well) were infected with negative con- trol (NC), or SIRT6 shRNA (si-SIRT6) were seeded in 96-well plates before being incubated at 37 ℃ for 1, 2, or
3 days. PBS was utilized in order to wash the cells. Every well received a supplement of 10 ul of MTT solution (5 mg/ ml) in order to achieve terminal concentration of 0.5 mg/ml and were incubated for 4 h. Subsequently, supernatants were removed and DMSO (100 ul) was added in order to dissolve formazan crystals. OD values were measured at 570 nm with the help of a plate reader (supplied by Multiskan GO, Thermo Fisher Scientific, America).
Cell cycle investigation
Cells underwent 12-h starvation in preparation of synchro- nization prior to re-stimulation via 10% FBS for 24 h. Cells underwent fixation with 75% ethanol and were processed in conformity with Cell Cycle Detection Kit (provided by BD Biosciences, Bedford, America). A FACS Caliber flow cytometer (provided by Beckman, America) was utilized in order to separate the cells. FlowJo software (offered by Tree- star Inc., America) was applied to evaluate the cell phase distributions.
Evaluation of cell apoptosis
Flow cytometry (FC) was carried out in order to assess cell death. Cells were washed twice with cold PBS, centrifuged for 5 min at 100 rpm, and resuspended in binding buffer. FITC-Annexin V and propidium iodide (PI) were added before incubating for 10 min at room temperature. Fluores- cence signals were evaluated using a FACScan flow cytome- ter (provided by Becton, Dickinson and Company, America).
TUNEL assay
H295R cells on the slide underwent fixation with the help of 4% paraformaldehyde (PFA). The In Situ Cell Death Detection Kit (provided by Roche) was utilized in order to conduct TUNEL labeling of dead cells as instructed by the manufacturer.
Cell migration assays
Transwell assays were applied to investigate migration. In short, 5 × 104 cells suspended in DMEM without serum including 1 µg/ml mitomycin C were seeded in wells at the top of a 24-well polycarbonate transwell filter (provided by Millipore, Bedford, America). DMEM including 10% serum was added to wells at the bottom. After a 24-h incubation, cells on the top of the filters were scraped off, while those at the bottom underwent fixation, staining, and quantification.
Cell invasion assay
Transwell chambers with Matrigel coating were used to evaluate cell invasion. Cells which underwent transfection (1.0×105 cells/chamber) were seeded at the top of the cham- ber and underwent a 24 h incubation at 5% CO2 and 37 ℃. As a chemoattractant, FBS (20%) was added to the chambers at the bottom. All cells without invasion at the top surface were eliminated via a cotton swab subsequent to incubation. Cells with invasion ability at the bottom surface underwent fixation with 100% methanol before staining with 1% crys- tal violet. Cells with invasion ability were quantified with a microscope, with 6 random fields selected per assay.
Western blot (WB) analysis
Radioimmunoprecipitation assay (RIPA) buffer was utilized in order to lyse the cells in the preparation of cell lysates. Proteins were quantified and dissolved with the help of 10% SDS-PAGE and were then transferred to immobilon poly- vinylidene difluoride (PVDF, 0.45 um) membranes. Five percent BSA was utilized in order to block the membranes for an hour at 25 ℃. Overnight incubation was carried out at 4C using the following antibodies: anti-CGRP (provided by Abcam, UK; ab47027), anti-TLR4 (offered by Abcam; ab22048), anti-p65 (provided by Abcam; ab16502), anti- TRPV1 (purchased from Thermo Fisher Scientific; PA1- 748), anti-SIRT6 (Abcam; ab105391), and anti-p-p65 (Abcam; ab28856). A 1-h incubation was carried out with secondary antibodies at 4 ℃. An enhanced chemilumines- cence reaction (Thermo Fisher Scientific; SuperSignal West Femto Maximum Sensitivity Substrate Kit) was utilized in order to observe the blots using a C-DiGit Blot Scanner (made in Shanghai, China).
Statistical analysis
Results are displayed as mean + SEM. Two-tailed, unequal- variance Student’s t test, or ANOVA prior to Tukey’s post hoc analysis were applied to assess the significance of dif- ferences between various groups. Values were considered statistically significant at P<0.05.
Results
SIRT6 expression was markedly decreased in adrenocortical carcinoma
In order to investigate SIRT6 expression profiles in colon cancer, our research first examined SIRT6 expression in eight matched ACC tissues as well as in normal tissues using WB, IHC, and RT-PCR. The expression of SIRT6 was
reduced in ACC tissues in comparison with normal tissues (Fig. 1). The findings above suggest that SIRT6 expression was inhibited in ACC.
SIRT6 knockdown increased proliferation of H295R human ACC cells
To investigate the role of SIRT6 in ACC cell proliferation, H295R cells were transfected with SIRT6-specific siRNA or with SIRT6 plasmids and cell proliferation were deter- mined by an MTT and FC assay. MTT assay results showed that proliferation of H295R cells was markedly increased after SIRT6 knockdown and was markedly decreased after SIRT6 over-expression compared with the negative con- trol (Fig. 2a, b). Flow cytometry analysis also showed that the H295R cells’ S phase was increased in samples treated with SIRT6-specific siRNA compared with negative con- trols (Fig. 2c, d). In addition, expression of the proliferative marker PCNA was enhanced in H295R cells after SIRT6 knockdown (Fig. 2e-g). These results indicate that SIRT6 knockdown increases H295R cell proliferation.
SIRT6 knockdown inhibited H295R human ACC cell apoptosis
To examine the role of SIRT6 in adrenocortical carcinoma cell apoptosis, the TUNEL assay was performed. Cells transfected with SIRT6-specific siRNA had a significantly decreased number of TUNEL positive cells when compared with the cells transfected with negative controls (Fig. 3a, b). The following FC utilizing an Annexin V/PI staining assay also confirmed the occurrence of cell apoptosis. The rate of apoptosis was markedly decreased in H295R cells after SIRT6 knockdown compared to NC (Fig. 3c, d). In addition, SIRT6 over-expression significantly increased the rate of cell apoptosis (Fig. 3 e-g). The findings above demonstrated that KD of SIRT6 inhibited apoptosis of adrenocortical carci- noma cells.
SIRT6 knockdown promoted H295R human ACC cell invasion and migration
We then examined the contribution of SIRT6 to invasive- ness as well as migration of ACC cells. Migration ability
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of H295R cells was examined via a migration assay with transwell chambers without coating. The results of the migration assay proved that SIRT6 KD significantly pro- moted migration (Fig. 4a, b). Invasion capability of H295R cells was evaluated via an invasion assay using polycar- bonate transwell filters which were preliminarily coated
with Matrigel. The quantity of H295R cells which invaded the Matrigel-precoated filters and arrived at bottom of the filter membrane was larger in H295R cells with SIRT6 KD than that of the NC (Fig. 4c, d). The findings above sug- gested that SIRT6 KD reinforced invasion and migration capabilities of H295R cells.
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SIRT6 KD enhanced the NF-KB signaling in H295R human ACC cells
Since NF-KB signaling pathways play a crucial role in adrenocortical carcinoma [19], we sought to explore the contribution of SIRT6 to stimulation of NF-KB signaling pathways. TLR4 was noticeably upregulated in SIRT6- specific siRNA-transfected H295R cells compared to nega- tive control-transfected H295R cells (Fig. 5). Moreover, the phosphorylation levels of IKK and p65 were enhanced more potently in SIRT6-specific siRNA-transfected H295R cells than in NC-transfected H295R cells. The data above
demonstrated that SIRT6 knockdown enhanced the NF-KB signaling in H295R cells.
SIRT6 knockdown increased the production of ROS and upregulates the TRPV1/CGRP pathway in H295R cells
Production of ROS was evaluated using a fluorescence probe, DCFH-DA. As shown in Fig. 6a, SIRT6 knockdown significantly increased ROS generation in H295R cells. In addition, the TRPV1 and CGRP level was increased in SIRT6-specific siRNA-transfected H295R cells compared to
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negative control-transfected H295R cells (Fig. 6b-d). These data suggested that SIRT6 knockdown enhanced the activa- tion of the TRPV1/CGRP pathway in H295R cells.
Discussion
In our research, SIRT6 was downregulated in ACC com- pared to matched noncancerous tissues. We also illustrated the function of SIRT6 in adrenocortical carcinoma cells using knockdown assays. The findings of this study revealed that SIRT6 knockdown promoted the cell proliferation, inhibited cell death, and migration as well as invasion abil- ity of ACC cells. Mechanistically, SIRT6 knockdown pro- motes the activation of the NF-KB signal pathway, increases generation of ROS and TRPV1/CGRP which then contrib- utes to enhanced proliferation and migration ability. Taken together, this study showed that SIRT6 is able to regulate the biological behaviors of adrenocortical carcinoma and that the NF-KB/TRPV1/CGRP pathway contributes to this process. Our findings provide novel insights not only in the contribution of SIRT6 to pathogenesis of ACC but also in the development of reagents for adrenocortical carcinoma treatment.
It was shown previously that SIRT6 expression was selectively inhibited in various human malignancies [20, 21], but excessive SIRT6 expression was also revealed in
osteosarcoma, hepatocellular carcinoma (HCC) as well as NSCLC [15, 22, 23]. Abnormal SIRT6 expression is com- monly recognized as an innovative predictor of clinical out- come of patients with cancer [24, 25]. Nevertheless, under- standing of SIRT6’s contribution to ACC is insufficient. It was discovered in our research that SIRT6 was downregu- lated in ACC in comparison with paired normal tissues. It was recently shown that SIRT6 KD elevated TRF2 concen- tration and promoted cell viability. It was also shown in our research that SIRT6 KD enhanced proliferation, invasion as well as migration capability of ACC cells. It was previously shown that excessive SIRT6 expression triggered the death of malignant cells instead of normal cells [26], which was in conformity with our research. It was shown via FC that SIRT6 KD inhibited cell death. It was previously proven that SIRT6 reinforces apoptosis in liver carcinogenesis in a diethylnitrosamine (DEN)-triggered murine liver cancer model [27]. Zhou et al. [24] demonstrated that SIRT6 over- expression inhibited proliferation as well as survival of gas- tric cancer cells.
The NF-KB pathway serves as a crucial regulator of cel- lular immune and stress reactions [28]. Abnormal NF-KB function has been identified in nearly every stage of tumori- genesis. Recent studies investigated direct or indirect partici- pation of this transcription factor system in cancer formation [29-31]. Although its indirect participation has an impact on nearly every cancer hallmark and exhibits malignant
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features, NF-KB is able to counteract these tumor promot- ing functions and consequently repressing global NF-KB inhibition [32, 33]. Direct participation could be attrib- utable to mutations in the NF-KB system [34, 35]. These mutations commonly take place in upstream agents, which causes stimulation of NF-KB separate from pathways that enhance malignancy generation [36, 37]. On the contrary, mutations in downstream agents, including DNA-binding subunits, participate in oncogenic transformation by exert- ing an impact on transcriptional output programs regulated
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by NF-KB [38, 39]. Consequently, NF-KB-modulating path- ways specific to certain cell types serve as promising tar- gets to treat malignancies. Our research proved that SIRT6 KD promoted NF-KB stimulation, increased expression of TRPV1/CGRP as well as ROS and subsequently brought about increased migration and proliferation capability of ACC cells.
In summary, SIRT6 serves as a tumor suppressor in ACC by regulating the NF-KB pathway, which could offer an innovative therapeutic target for ACC treatment.
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Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval This study has been approved by the Ethics Commit- tee of the West China Hospital, Sichuan University.
Informed consent Informed consent was obtained from all individual participants included in the study.
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